Streptomyces coelicolor is the prototypic Streptomycete spp. Its physiological and genetic properties have been studied extensively. The genome of this soil bacterium contains 18 cytochrome P450s (CYP) genes, 6 ferredoxin genes and 4 ferredoxin reductase genes. This complex of monooxygenase systems plays important roles in the biosynthesis of secondary metabolites, organic molecules which serve several different roles in S. coelicolor, particularly protection against toxic organisms and compounds in the environment. Many of these compounds are known to have biomedical activity and are used in treatment of human disease. The P450s are not essential for primary metabolism in this and other streptomycetes but oxidatively tailor secondary metabolites influencing both potency and specificity. Engineering of these P450s can lead to novel compounds having new biomedical capacity and the goal of this project is to develop a biochemical paradigm for generating new metabolites. During the current granting period we have developed the necessary technology and obtained preliminary data which will permit us to move aggressively forward in addressing this goal. This project involves two laboratories at Vanderbilt (Waterman and Guengerich) and two at the University of Wales, now Swansea University (Kelly and Lamb), and three Specific Aims. (1) Determination of Function of Streptomyces P450s - will be carried out using prediction of substrate based on location of CYP genes in operons having known activity in cells, or based on similarity of primary sequence with P450s of known function, and use of knockout strains for analysis of differences in metabolite profiles compared to the wild type strain. These differences are measured using sophisticated GC-MS and LC-MS technologies. (2) Structure Determination of Streptomyces P450s and Biophysical Investigation of Novel P450 Properties -we will focus our effort of CYP X-ray structure determination primarily on P450s having known functions to characterize how substrates bind in the active sites. (3) Generation of Novel Secondary Metabolites in S. coelicolor - putting together our understanding of CYP function with that of CYP structure we will generate novel secondary metabolites relying on site directed mutagenesis, product analysis, expression of mutant enzymes in S. coelicolor, and additional structure determination (if necessary). During the 40 months of the current funding period we have been surprised to find several novel features of these P450s (described within) which expand our understanding of the diversity of this superfamily of monooxygenases. As part of Aim 2 we will further investigate these properties. The significance of these three Specific Aims is that they will develop a paradigm for engineering novel streptomyces secondary metabolites with the potential of important new biomedical relevance and will provide new insights into cytochrome P450 structure/function. PUBLIC HEALTH RELEVANCE: This competing renewal of "S. coelicolor P450s: Structure/Function/Engineering" will continue ongoing studies of P450s in a well characterized soil bacterium with the long term goal to develop a paradigm for using modification of these monooxygenases to generate novel biomedically relevant secondary metabolites. Actinomycetes (including S. coelicolor) produce a large number of different secondary metabolites many of which have biomedical relevance. In many cases P450s oxidatively tailor these molecules near the end of their biosynthetic pathways and modification of P450s can lead to new compounds having novel potency and specificity.